Establishment Of A Mouse Model Of Chimeric PCV1-2b And Preliminary Exploration On Attenuated Chimeric PCV1-2b Vaccine Triggering Immune Response In Pigs

Porcine circovirus(PCV)belongs to the family Circoviridae,genus Circovirus,which is the smallest circular DNA virus of animal known in the world.By genotyping,PCV can be divided into PCV1,PCV2 and PCV3.What’s more.PCV2 can be divided into PCV2a,PCV2b.PCV2c,PCV2d,PCV2e and PCV2f subtypes,recently,PCV2b and PCV2d are prevalent in China.It had been proved that PCV1 is not pathogenic,and PCV2 is the main cause of porcine circovirus-associated disease(PCVAD),which can lead to the postweaning multisystemic wasting syndrome(PMWS),porcine dermatitis and nephropathy syndrome(PDNS),porcine respiratory disease complex(PRDC)and reproductive system diseases.Recently,PCV3 has been reported to be associated with PDNS-like clinical signs,reproductive failure,cardiac pathologies and multisystemic inflammation in piglets and sows.Currently,some vaccines include inactivated PCV2 whole virus vaccines,inactivated recombinant virus vaccines,recombinant subunit vaccines were developed or commercially available,all of them are inactivated vaccines The attenuated chimeric PCV1-2b can produce good cross-immunization protection against PCV2b and PCV2d originated in China,which is an important candidate vaccine for PCV2 prevention in the future1.Establishment of a mouse model of chimeric PCV1-2bThe attenuated chimeric PCV1-2b vaccine and a PCV2b isolate were used to vaccinate eighteen BALB/c mice,respectively,other eighteen mice were used as mock.During the experiment,three mice from each group were randomly selected for necroscopy weekly,the DNA copies in serum and tissues were quantified by quantitative polymerase chain reaction(qPCR);The specific antibodies in the serum of the tested mice were monitored by indirect immunofluorescence assay(IFA).Interferon-γ-secreting cells(IFN-y-SCs),interleukin-2(IL-2),IL-4,and IL-10 in the supernatants of spleen lymphocytes(SLCs)from the inoculated mice cultured in vitro were monitored by enzyme-linked immunospot assay(ELISPOT)and enzyme-linked immunosorbent assay(ELISA),respectively.Results showed that viremia was first detected at 7 d.p.i.and then declined persistently.DNA copies in the tissues were the same in the two inoculation groups,except in the liver,where they were higher in PCV2b-inoculated group.PCV1-2b elicited a specific humoral response with greater antibody production than the PCV2b isolate.IFN-γ-SCs were clearly upregulated at 14,21,28,35 and 42 d.p.i.with either PCV1-2b or PCV2b.IL-2 was upregulated at 21 and 28 d.p.i.in the PCV1-2b-inoculated group and at 35 d.p.i.in the PCV2b-inoculated group.In both the PCV1-2b-and PCV2b-inoculated mice,IL-4 were always below the limit of detection and IL-10 was detected of low levels during experiment.2.Preliminary exploration on attenuated chimeric PCVl-2b vaccine triggering immune response in pigsOur previous studies showed that the attenuated chimeric PCV1-2b vaccine can provide cross-protection in pigs against the both PCV2b and PCV2d strains challenge,and a dose of 1×103.5TCID50 per animal of the vaccine can protect the pigs against either PCV2b or PCV2d strain challenge fully.To explore the immune efficacy of PCV1-2b,especially to compare the difference between pig and mouse in the humoral and cell-mediated immunity,five 21-day-old weaned piglets were inoculated with PCV1-2b and PCV2b respectively,additional five were used as mock.At 0,7,14,21,28,35 and 42 d.p.i.,the serum of tested piglets were used to detect the concentrations of interleukin-2(IL-2),IL-4,IL-10 and PCV2-specific antibodies by enzyme-linked immunosorbent assay(ELISA)and indirect immunofluorescence assay(IFA),interferon-γ-secreting cells(IFN-γ-SCs)in peripheral blood mononuclear cells(PBMCs)of piglets were counted by enzyme-linked immunospot assay(ELISPOT).In addition,the clinical status and average daily gain of the tested piglets were recorded.Moreover,viremia was also assessed.The results showed that the virus replications were comparable in the PCV1-2b-and PCV2b-inoculated piglets’ blood.From 28 to 42 d.p.i.,PCV1-2b could elicit greater antibodies than PCV2b.IFN-γ-SCs in PCV1-2b-inoculated pigs were significantly higher than those in the PCV2b-and the mock-inoculated pigs at 28 d.p.i..At 28 and 35 d.p.i.,IL-2 level was also significantly higher in PCV1-2b-inoculated pigs than mock pigs.There was no significant difference in IL-4 level among the three groups.IL-10 in PCV2b-inoculated pigs increased slightly in the early stage of the experiment(0-21 d.p.i.),but there was not any obviously change in PCV1-2b-and mock-inoculated group.